Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.
